General Introduction to Problem Area
Somatic embryogenesis in plants is a process in which somatic embryos are formed from an initial explant being a cell in a plant tissue. The somatic embryos formed are genetically identical copies of the plant providing the initial explant. The process of somatic embryogenesis thereby offers a tool to obtain large numbers of genotypically identical plants for multiplication of selected genotypes of commercial interest, for conservation of endangered species or for generating genetically uniform plant material for research purposes.
Physiological Background to the Procedures Related to the Problem
To produce plants from somatic embryos of conifers, a multi-step procedure is applied to meet the physiological needs of the different stages of development as described below and shown in FIG. 1. Initiation of somatic embryogenesis starts with induction of somatic embryos from an initial explant, typically an immature zygotic embryo, on a solidified culture medium containing plant growth regulator. Somatic embryos continue to form, typically on the same composition culture medium, and a proliferating embryogenic culture form. At the proliferating stage, several of the key features generally regarded as beneficial for the process of somatic embryogenesis process, take place: (i) the mass propagation of genotypically identical propagules through unlimited multiplication of immature somatic embryos; (ii) cryogenic storage of proliferating embryos substantiates an virtually eternal store of clones, i.e. a clone bank is established, (iii) transgenic modification of the immature somatic embryo allow for large scale propagation of genetically improved propagules. At the next step in the procedure, the proliferating somatic embryo is subjected to a growth medium that triggers embryo development to progress into the maturation stage. Conversion from proliferation to maturation only occurs in a fraction of the proliferating embryos in the culture. Low conversion rates are encountered more frequently in genotypes from recalcitrant conifer species, but are common in all conifer species as well as other plant species. The manual labour needed to collect embryos increase with the decrease in conversion rate, and thereby the cost and risk of contamination and other inaccuracies. Low conversion rate from proliferation to maturation is a major bottleneck for commercial large scale applications of somatic embryogenesis procedures. For germination, mature somatic embryos are subjected to different culture regimes to induce root- and shoot formation, in as number of different steps; desiccation, sucrose treatment, red tight, induction, and blue light stimulation. Thereafter, germinated embryos deemed appropriately developed are transferred to a compost material and gradually transferred to an environment ex vitro during winch the sucrose content is reduced. The different treatments during germination into a plant requires repeated manual handling of individual germinants and plants adding a considerable cost to the overall procedure.
Production of Plants from Somatic Embryos
The prior art procedure for producing plants from somatic embryos requires manual handling at several steps making the procedure time consuming, expensive and inaccurate.
For conifer species, standard procedures used involve several steps when manual handling is required. The general procedure is outlined in FIG. 1 (sec e.g. von Arnold S. Clapham D, Spruce embryogenesis. 2008. Methods Mol Biol. 2008; 427:31-47; Belmonte M F, Donald G. Reid D M. Yeung E C and Stasolla C. 2005. Alterations of the glutathione redox state improve apical mainstem structure and somatic embryo quality in white spruce (Picea glauca), J Exp Biol, Vol. 56, No. 419, pp, 2355-2364).
There are four steps that rely on manual handling to obtain a small plant from the mature somatic embryo as seen in FIG. 1. The first manual interaction is when [1] the mature embryo is isolated from immature embryos (120), and placed horizontally in a plastic container under sterile conditions; the second [2] occur after 3-7 days of resting (130), then mature embryo is transferred to a gelled culture medium for initiation of germination processes. The germinated somatic embryo will under appropriate culture medium composition and light conditions initiate roots (140). The third manual transfer [3] is when the germinant having a small root formed is transferred to an upright position with the root partially immersed in liquid germination media (150). The fourth [4] and final transfer is when the germinated embryos has a to root and small lateral roots, then it is transferred into a solid substrate in a pot for further plant formation (160).
TABLE 1List of designations pertaining to FIG. 1.ItemDesignation100Mature embryo101Crown of a mature embryo102Foot of a mature embryo103Width of crown of a mature embryo104Length of a mature embryo120Maturation phase130Resting phase140Germination phase150In vitro plant formation phase160Ex vitro plantformation phase
Conversion from proliferation to maturation only occurs in a fraction of the proliferating embryos in the culture. Low conversion rates are encountered more frequently in genotypes from recalcitrant conifer species, but are common in all conifer species as well as other plant species. The manual labour needed to collect embryos increase with the decrease in conversion rate, and thereby the cost and risk of contamination and other inaccuracies. Low conversion rate from proliferation to maturation is a major bottleneck for commercial large scale applications of somatic embryogenesis procedures. For germination, mature somatic embryos are subjected to different culture regimes to induce root- and shoot formation, in a number of different steps; desiccation, sucrose treatment red light induction, and blue light stimulation. Thereafter germinated embryos deemed appropriately developed arc transferred to a compost material and gradually transferred to an environment ex vitro during which the sucrose content is reduced. The different treatments during Remittal into a plant requires repeated manual handling of individual germinants and plants adding a considerable cost to the overall procedure.
In the hitherto available method for producing plants from somatic embryos the embryos are picked out manually from the immature embryogenic tissue is time-consuming and ineffective. It would therefore be desirable to provide a way to make the separation of the embryos more effective. The somatic embryos produced are daily glued together by immature embryogenic tissue into clusters. It is an object of the invention to provide effective means of producing somatic plant embryos, an automated means for gently dispersing the clusters of somatic embryos into individual embryos detached from the embryogenic tissue. The invention relates to a method and a device for such dispersion.